This invention relates to the field of genetically engineered, redirected immune cells and to the field of cellular immunotherapy of B-cell malignancies, B-cell lymphoproliferative syndromes and B-cell mediated autoimmune diseases. The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice are incorporated by reference.
Approximately half of all hematopoietic stem cell transplantation (HSC) procedures performed in the United States are for the treatment of hematologic malignancy [1]. The initial obstacles for successful HSC transplantation were in large part due to inadequate treatment modalities for ameliorating regimen-related toxicities and for controlling opportunistic infections and graft-versus-host disease (GVHD) [2-5]. As supportive care measures have improved over the last decade, post-transplant disease relapse has emerged as the major impediment to improving the outcome of this patient population [6-10]. The inability of maximally intensive preparative regimens combined with immunologic graft-versus-tumor reactivity to eradicate minimal residual disease is the mechanism of treatment failure in allogeneic transplantation while, in the autologous setting, tumor contamination of the stem cell graft can also contribute to post-transplant relapse [11]. Targeting minimal residual disease early after transplantation is one strategy to consolidate the tumor cytoreduction achieved with myeloablative preparative regimens and purge, in vivo, malignant cells transferred with autologous stem cell grafts. The utility of therapeutic modalities for targeting minimal residual disease shortly following stem cell rescue is dependent on both a limited spectrum of toxicity and the susceptibility of residual tumor cells to the modality's antitumor effector mechanism(s). The successful elimination of persistent minimal residual disease should not only have a major impact on the outcome of transplantation for hematologic malignancy utilizing current myeloablative preparative regimens but may also provide opportunities to decrease the intensity of these regimens and their attendant toxicities.
The prognosis for patients with bcr-abl positive Acute Lymphoblastic Leukemia (ALL) treated with chemotherapy is poor and allogeneic transplantation has offered a curative option for many patients when an appropriate donor was available. For example at the City of Hope, 76 patients with bcr-abl positive ALL were treated with allogeneic Bone Marrow Transplantation (BMT) from a HLA matched donor. Of these patients, 26 were in first remission, 35 were transplanted after first remission. The two year probability of disease free survival was 68% with a 10% relapse rate in those patients transplanted in first remission whereas for those patients transplanted after first remission, the disease-free survival and relapse rate were 36% and 38%, respectively [12]. Post-transplant Polymerase Chain Reaction (PCR) screening of blood and marrow for bcr-abl transcript is under evaluation as a molecular screening tool for identifying early those transplant recipients at high risk for later development of overt relapse [13,14]. Patients for whom detectable p190 transcript was detected following BMT had a 6.7 higher incidence of overt relapse than PCR negative patients. The median time from the development of a positive signal to morphologic relapse was 80-90 days in these patients. The identification of patients in the earliest phases of post-transplant relapse affords the opportunity for making therapeutic interventions when tumor burden is low and potentially most amenable to salvage therapy.
Recent advances in the field of immunology have elucidated many of the molecular underpinnings of immune system regulation and have provided novel opportunities for therapeutic immune system manipulation, including tumor immunotherapy. Evidence supporting the potential of immune-mediated eradication of residual tumor cells following allogeneic transplantation can be inferred by comparing the disparate relapse rates between recipients of syngeneic and non-T cell depleted matched sibling transplants. Patients with chronic myelogenous leukemia in chronic phase (CML-CP), acute myelogenous leukemia in first complete remission (1st CR), and acute lymphoblastic leukemia in 1st CR who received a marrow transplant from a syngeneic donor had an actuarial probability of relapse at 3 years of 45%, 49%, and 41%, respectively, whereas the rates for recipients of a non-T depleted marrow transplant from an HLA identical sibling for the same diseases were 12%, 20%, and 24%, respectively [15-17]. The reduction of relapse rates following allogeneic bone marrow transplantation has been most significant in patients who develop acute and/or chronic GVHD. Currently, efforts are focused on developing strategies to selectively augment the graft-versus-leukemia (GVL) response in order to reduce post-transplant relapse rates without the attendant toxicities of augmented GVHD.
Studies in animal models have established that donor MHC-restricted CD8+ and CD4+ α/β+ T cells specific for minor histocompatibility antigens encoded by polymorphic genes that differ between the donor and recipient are the principle mediators of acute GVHD and GVL [18-21]. Recently, patients with CML in chronic phase who relapse after allogeneic BMT have been identified as a patient population for whom the infusion of donor lymphocytes (DLI) successfully promotes a GVL effect [22,23]. Complete response rates of approximately 75% are achieved with DLI cell doses in the range of 0.25-12.3×108 mononuclear cells/kg [24]. Although the antitumor activity of donor lymphocyte infusion underscores the potential of cellular immunotherapy for CML, the clinical benefit of DLI has not been generalizable to all forms of hematologic malignancy. Relapsed ALL is much less responsive to DLI with a reported CR rate of less than 20%; when tumor responses are observed, they are typically associated with significant GVHD morbidity and mortality [25]. In order to increase the therapeutic ratio of DLI, genetic modification of donor lymphocytes to express a suicide gene is being evaluated as a strategy to permit the in vivo ablation of donor lymphocytes should toxicity from GVHD warrant this maneuver [26,27]. Alternately, efforts are underway to identify genes encoding minor histocompatibility antigens (mHA's) with restricted hematopoietic expression that elicit donor antigen-specific T cell responses. The isolation, ex vivo expansion, and re-infusion of donor-derived clones specific for these mHA's has the potential of selectively augmenting GVL following allogeneic bone marrow transplantation [28-30].
Non-transformed B-cells and malignant B-cells express an array of cell-surface molecules that define their lineage commitment and stage of maturation. These were identified initially by murine monoclonal antibodies and more recently by molecular genetic techniques. Expression of several of these cell-surface molecules is highly restricted to B-cells and their malignant counterparts. CD20 is a clinically useful cell-surface target for B-cell lymphoma immunotherapy with anti-CD20 monoclonal antibodies. This 33-kDa protein has structural features consistent with its ability to function as a calcium ion channel and is expressed on normal pre-B and mature B cells, but not hematopoietic stem cells nor plasma cells [31-33]. CD20 does not modulate nor does it shed from the cell surface [34]. In vitro studies have demonstrated that CD20 crosslinking by anti-CD20 monoclonal antibodies can trigger apoptosis of lymphoma cells [35,36]. Clinical trials evaluating the antitumor activity of chimeric anti-CD20 antibody IDEC-C2B8 (Rituximab) in patients with relapsed follicular lymphoma have documented tumor responses in nearly half the patients treated, although the clinical effect is usually transient [37-40]. Despite the prolonged ablation of normal CD20+ B-cells, patients receiving Rituximab have not manifested complications attributable to B-cell lymphopenia [41]. Radioimmunotherapy with 131I-conjugated and 90Y-conjugated anti-CD20 antibodies also has shown promising clinical activity in patients with relapsed/refractory high-grade Non-Hodgkins Lymphoma but hematopoietic toxicities from radiation have been significant, often requiring stem cell support [42].
Unlike CD20, CD19 is expressed on all human B-cells beginning from the initial commitment of stem cells to the B lineage and persisting until terminal differentiation into plasma cells [43]. CD19 is a type I transmembrane protein that associates with the complement 2 (CD21), TAPA-1, and Leu13 antigens forming a B-cell signal transduction complex. This complex participates in the regulation of B-cell proliferation [44]. Although CD19 does not shed from the cell surface, it does internalize [45]. Accordingly, targeting CD19 with monoclonal antibodies conjugated with toxin molecules is currently being investigated as a strategy to specifically deliver cytotoxic agents to the intracellular compartment of malignant B-cells [46-48]. Anti-CD19 antibody conjugated to blocked ricin and poke-weed antiviral protein (PAP) dramatically increase specificity and potency of leukemia cell killing both in ex vivo bone marrow purging procedures and when administered to NOD-SCID animals inoculated with CD19+ leukemia cells [49]. In vitro leukemia progenitor cell assays have provided evidence that the small percentage of leukemic blasts with the capacity for self-renewal express CD19 on their cell surface. This conclusion was derived from the observations that leukemic progenitor activity is observed exclusively in fresh marrow samples sorted for CD19 positive cells and is not observed in the CD19 negative cell population [50]. Additionally B43-PAP treatment of relapsed leukemic marrow specimens ablates progenitor cell activity while a PAP conjugated antibody with an irrelevant specificity had no such activity [51]. Systemic administration of the CD19-specific immunotoxin B43-PAP is currently undergoing investigation in phase I/II clinical trials in patients with high risk pre-B ALL [52].
Despite the antitumor activity of monoclonal anti-CD20 and anti-CD19 antibody therapy observed in clinical trials, the high rate of relapse in these patients underscores the limited capacity of current antibody-based immunotherapy to eliminate all tumor cells [53]. In contrast, the adoptive transfer of tumor-specific T cells can result in complete tumor eradication in animal models and a limited number of clinical settings [54,55]. The ability of transferred T cells to directly recognize and lyse tumor targets, produce cytokines that recruit and activate antigen non-specific antitumor effector cells, migrate into tumor masses, and proliferate following tumor recognition all contribute to the immunologic clearance of tumor by T cells [56]. Expression-cloning technologies have recently permitted the genetic identification of a growing number of genes expressed by human tumors to which T cell responses have been isolated [57,58]. To date leukemia and lymphoma-specific antigens have not been identified that are both broadly expressed by malignant B-cells and elicit T cell responses. Consequently, preclinical and clinical investigation has focused on combining antibody targeting of tumors with T cell effector mechanisms by constructing bispecific antibodies consisting of CD20 or CD19 binding sites and a binding site for a cell-surface CD3 complex epitope. Such bispecific antibodies can co-localize leukemia and lymphoma targets with activated T cells resulting in target cell lysis in vitro [59-61]. The in vivo antitumor activity of such bispecific antibodies has been limited, however, both in animal models as well as in clinical practice [62]. The discrepancy between in vitro activity and in vivo effect likely reflects the inherent limitations in antibody immunotherapy compounded by the obstacles associated with engaging T cells and tumor cells via a soluble linker in a manner that yields a persistent and functional cellular immune response [63].
The safety of adoptively transferring antigen-specific CTL clones in humans was originally examined in bone marrow transplant patients who received donor-derived CMV-specific T cells [56]. Previous studies have demonstrated that the reconstitution of endogenous CMV-specific T cell responses following allogeneic bone marrow transplantation (BMT) correlates with protection from the development of severe CMV disease [64]. In an effort to reconstitute deficient CMV immunity following BMT, CD8+ CMV-specific CTL clones were generated from CMV seropositive HLA-matched sibling donors, expanded, and infused into sibling BMT recipients at risk for developing CMV disease. Fourteen patients were treated with four weekly escalating doses of these CMV-specific CTL clones to a maximum cell dose of 109 cells/m2 without any attendant toxicity [65]. Peripheral blood samples obtained from recipients of adoptively transferred T cell clones were evaluated for in vivo persistence of transferred cells. The recoverable CMV-specific CTL activity increased after each successive infusion of CTL clones, and persisted at least 12 weeks after the last infusion. However, long term persistence of CD8+ clones without a concurrent CD4+ helper response was not observed. No patients developed CMV viremia or disease. These results demonstrate that ex-vivo expanded CMV-specific CTL clones can be safely transferred to BMT recipients and can persist in vivo as functional effector cells that may provide protection from the development of CMV disease.
A complication of bone marrow transplantation, particularly when marrow is depleted of T cells, is the development of EBV-associated lymphoproliferative disease [66]. This rapidly progressive proliferation of EBV-transformed B-cells mimics immunoblastic lymphoma and is a consequence of deficient EBV-specific T cell immunity in individuals harboring latent virus or immunologically naive individuals receiving a virus inoculum with their marrow graft. Clinical trials by Rooney et al. have demonstrated that adoptively transferred ex-vivo expanded donor-derived EBV-specific T cell lines can protect patients at high risk for development of this complication as well as mediate the eradication of clinically evident EBV-transformed B cells [54]. No significant toxicities were observed in the forty-one children treated with cell doses in the range of 4×107 to 1.2×108 cells/m2.
Genetic modification of T cells used in clinical trials has been utilized to mark cells for in vivo tracking and to endow T cells with novel functional properties. Retroviral vectors have been used most extensively for this purpose due to their relatively high transduction efficiency and low in vitro toxicity to T cells [67]. These vectors, however, are time consuming and expensive to prepare as clinical grade material and must be meticulously screened for the absence of replication competent viral mutants [68]. Rooney et al. transduced EBV-reactive T cell lines with the NeoR gene to facilitate assessment of cell persistence in vivo by PCR specific for this marker gene [69]. Riddell et al. have conducted a Phase I trial to augment HIV-specific immunity in HIV seropositive individuals by adoptive transfer using HIV-specific CD8+ CTL clones [70]. These clones were transduced with the retroviral vector tgLS+HyTK which directs the synthesis of a bifunctional fusion protein incorporating hygromycin phosphotransferase and herpes virus thymidine kinase (HSV-TK) permitting in vitro selection with hygromycin and potential in vivo ablation of transferred cells with gancyclovir. Six HIV infected patients were treated with a series of four escalating cell dose infusions without toxicities, with a maximum cell dose of 5×109 cells/m2 [70].
As an alternate to viral gene therapy vectors, Nabel et al. used plasmid DNA encoding an expression cassette for an anti-HIV gene in a Phase I clinical trial. Plasmid DNA was introduced into T cells by particle bombardment with a gene gun [71]. Genetically modified T cells were expanded and infused back into HIV-infected study subjects. Although this study demonstrated the feasibility of using a non-viral genetic modification strategy for primary human T cells, one limitation of this approach is the episomal propagation of the plasmid vector in T cells. Unlike chromosomally integrated transferred DNA, episomal propagation of plasmid DNA carries the risk of loss of transferred genetic material with cell replication and of repetitive random chromosomal integration events.
Chimeric antigen receptors engineered to consist of an extracellular single chain antibody (scFvFc) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (ζ) have the ability, when expressed in T cells, to redirect antigen recognition based on the monoclonal antibody's specificity [72]. The design of scFvFc:ζ receptors with target specificities for tumor cell-surface epitopes is a conceptually attractive strategy to generate antitumor immune effector cells for adoptive therapy as it does not rely on pre-existing anti-tumor immunity. These receptors are “universal” in that they bind antigen in a MHC independent fashion, thus, one receptor construct can be used to treat a population of patients with antigen positive tumors. Several constructs for targeting human tumors have been described in the literature including receptors with specificities for Her2/Neu, CEA, ERRB-2, CD44v6, and epitopes selectively expressed on renal cell carcinoma [73-77]. These epitopes all share the common characteristic of being cell-surface moieties accessible to scFv binding by the chimeric T cell receptor. In vitro studies have demonstrated that both CD4+ and CD8+ T cell effector functions can be triggered via these receptors. Moreover, animal models have demonstrated the capacity of adoptively transferred scFvFc:ζ expressing T cells to eradicate established tumors [78]. The function of primary human T cells expressing tumor-specific scFvFc:ζ receptors have been evaluated in vitro; these cells specifically lyse tumor targets and secrete an array of pro-inflammatory cytokines including IL-2, TNF, IFN-γ, and GM-CSF [79]. Phase I pilot adoptive therapy studies are underway utilizing autologous scFvFc:ζ-expressing T cells specific for HIV gp120 in HIV infected individuals and autologous scFvFc:ζ-expressing T cells with specificity for TAG-72 expressed on a variety of adenocarcinomas including breast and colorectal adenocarcinoma.
Investigators at City of Hope have engineered a CD20-specific scFvFc:ζ receptor construct for the purpose of targeting CD20+ B-cell malignancy [80]. Preclinical laboratory studies have demonstrated the feasibility of isolating and expanding from healthy individuals and lymphoma patients CD8+ CTL clones that contain a single copy of unrearranged chromosomally integrated vector DNA and express the CD20-specific scFvFc:zζ receptor [81]. To accomplish this, purified linear plasmid DNA containing the chimeric receptor sequence under the transcriptional control of the CMV immediate/early promoter and the NeoR gene under the transcriptional control of the SV40 early promoter was introduced into activated human peripheral blood mononuclear cells by exposure of cells and DNA to a brief electrical current, a procedure called electroporation [82]. Utilizing selection, cloning, and expansion methods currently employed in FDA-approved clinical trials at the FHCRC, gene modified CD8+ CTL clones with CD20-specific cytolytic activity have been generated from each of six healthy volunteers in 15 separate electroporation procedures [81]. These clones when co-cultured with a panel of human CD20+ lymphoma cell lines proliferate, specifically lyse target cells, and are stimulated to produce cytokines.
It is desired to develop additional redirected immune cells and, in a preferred embodiment, redirected T cells, for treating B-cell malignancies and B-cell mediated autoimmune disease.